Calcium is one of the more important elements found in the body. It is necessary not only for the skeleton but also for cells. There is, on average, about one kilogram of calcium in the human body of which 99% is located in bone with the remaining 1% distributed in plasma, extracellular fluids and intercellular compartments. This small fraction, however, plays a vital role in many biochemical and physiological functions such as a cell regulator and messenger. These functions include bone formation and homostasis, maintenance of cell membrane integrity and permeability, nerve excitation, muscular contraction and blood coagulation together with regulation of many enzyme and hormone reactions.
The concentration of calcium in body fluids, particularly in plasma, needs to be kept within a very narrow range. Its level is controlled by a number of hormones, primarily by parathyroid hormone (PTH), and calcitonin. PTH is released from the parathyroid gland in response to a decrease of calcium concentration in plasma and indirectly promotes calcium absorption in the intestine and renal tubules and increases the calcium mobilization from bone. Calcitonin, which inhibits PTH activity in bone tissue, is secreted by the thyroid gland in response to a rise in calcium ion.
Deviations from normal calcium levels occur in certain diseases. Calcium levels significantly less than normal can be indicative of hypoparathyroidism, Vitamin D deficiency or nephritis. Calcium levels of greater than normal may indicate hyperparathyroidism, Vitamin D intoxication or myeloma.
The normal value of total calcium in plasma is about 2.4 mM/L. Generally, infants have the highest calcium concentration which declines slightly with age.
The determination of calcium in serum began with the gravimetric method in which calcium was precipitated with ammonium oxalate whereupon the precipitate was dried and weighed. This method was improved upon in 1921 when there was reported a technique in which the calcium oxalate is dissolved in acid with the oxalate being determined by titration with potassium permanganate. A modification of this method, in which the washing procedure and temperature were standardized during the titration, was used as the primary procedure for calcium determination. While reasonably accurate, these procedures required large amounts of serum and were time consuming. A more sensitive and rapid complexometric titration was introduced in the 1940's which used murexide as an indicator. Several other indicators, e.g. calcon, calcein, methylthymol blue, eriochrome black T, glyoxal bis-(2-hydroxyanil) and arsenazo III, were subsequently introduced. Regardless of what indicator was used, these complexometric titration methods required a large volume of serum sample, were time consuming and suffered from a poor end-point as well as interferences by metal ions other than calcium.
More recently, the titration method was replaced by a direct spectrophotometric method using various metallochromic indicators, the most popular of which is the ortho-cresolphthalein (CPC) complex method. In this method, calcium combines with CPC in an alkaline solution (pH 10.5 to 12) to form a deep purple calcium-dye complex. The dye's absorbance increases at 575 nm and is proportional to the concentration of calcium in the sample. A disadvantage of this method is the requirement that it be carried out at a pH in the 10 to 12 range. At this pH level, the reagent can absorb carbon dioxide resulting in baseline drift.
Arsenazo III forms colored complexes with many divalent and trivalent cations but can be used to determine micromolar quantities of calcium ion at pH 5.5 without significant interference from magnesium ion. This reagent has a high affinity for calcium ion at the physiological pH, a high extinction coefficient of the calcium-dye complex at 650 nm and exhibits high chemical stability in aqueous solutions. Accordingly it has become a useful tool for determining micromolar concentrations of calcium in single cells. While arsenazo III is widely used by researchers in studies of calcium transport in cells and cell fractions, its utility in clinical chemistry has been limited due to the presence of toxic arsenic moieties and their concomitant safety and environmental concerns.
In Biochemistry 19, 2396 (1980) Tsien reports the preparation of 2-[[2-bis(ethoxycarbonyl)methyl]amino]-quinoline(QUIN1) and its 6-methoxy analog (QUIN2). These compounds are described as having utility as fluorescent calcium ionophores. In a later publication, Tsien et al. describe monitoring the fluoroscence of QUIN2 as being the most popular method for measuring [Ca.sup.++ ]. They go on to point out that, while QUIN2 has revealed much important biological information, its use has some inherent limitations since its preferred excitation wavelength of 339 nm is too low. It is also pointed out that its extinction coefficient (&lt;5000) and fluorescence quantum yield (0.03 to 0.14) are also too low. In addition, autofluorescence from cells requires QUIN2 loadings of several tenths millimolar or more to obtain a satisfactory result. It is also pointed out that QUIN2 signals Ca.sup.++ by increasing its fluorescence intensity without much shift in either excitation or emission wavelengths and that there is a need for an indicator which responds to calcium by shifting wavelengths while maintaining strong fluorescence. Another deficiency reported for QUIN2 is that its selectivity for calcium over magnesium and heavy metal divalent cations could bear improvement. This article goes on to point out that compounds having a stilbene fluorophore and an octacoordinate, tetracarboxylate pattern of liganding groups characteristic of EGTA, [(ethylene glycol bis(.beta.-aminoethyl ether)] and BAPTA, [1,2-bis (o-aminophenoxy) ethanol-N,N,N'N'-tetraacetic acid] are preferable to QUIN2. This preference is based on several factors such as improved selectivity for Ca.sup.++ and the ability of BAPTA and EGTA to exhibit much stronger fluorescence together with wavelength shifts upon Ca.sup.++ binding. The preparation and utility of these compounds is also disclosed in U.S. Pat. 4,603,209 to Tsien et al.
More recently Toner et al. have disclosed chromogenic derivatives of BAPTA and BAPTA like compounds in U.S. Pat. No. 4.795,712. They point out that the fluorogenic compounds of Tsien suffer from the disadvantage of adsorbing in the ultraviolet region of the spectrum, so that normal constituents of body fluids which also adsorb in the UV and short visible wavelengths tend to produce background interference with standard colorimetric equipment and procedures. They go on to say that it would be desirable to have highly selective calcium complexing compounds which would be detectable at longer wavelengths (above 400 nm) and would shift to other wavelengths when complexed with calcium to allow quantitative analysis for calcium without interference from UV and short wavelength visible light-absorbing species.
The present invention is predicated on the discovery that arylazo derivatives of QUIN1 and QUIN2 can be effectively used for the colorimetric determination of Ca.sup.++ since they are highly selective for calcium in media which also contains magnesium ion. Furthermore, these compounds adsorb light at longer wavelengths than do similarly derivatized BAPTAs and exhibit a significantly greater shift in the maximum absorbance of the complexed -vs- non-complexed compound than do the corresponding chromogenic BAPTA compounds.